Transfection of Lens Cells with Phakinin Mutants

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DESCRIPTION (provided by applicant): The long-term objectives of the proposed studies are to determine how the lens-specific beaded filament protein (BFP) cytoskeleton contributes to lenticular clarity. Our overlying hypothesis is that recently identified mutations in the BF subunit phakinin associated with human cataract formation interrupt BF self-assembly or biochemical processing. Specific Aim 1 will test the hypothesis that the in situ biochemical characteristics of BFPs are not altered under in vitro conditions or during the transfection of cultured cells with BF subunit constructs fused to marker protein tags. This will be accomplished with confocal microscopy and immunochemical detection in cultured chicken lens annular pad cells transfected with BF subunit constructs fused to fluorescent proteins (FPs) or the HA epitope. Specific Aim 2 will test the hypothesis that phakinin mutations associated with human cataracts interfere with the polymerization and/or biochemical processing of BFPs during in vitro lens fiber differentiation. This will be accomplished in cultured cells transfected with mutated phakinin subunits fused to fluorescent proteins or antigenic determinants. BF polymerization and biochemical processing will be monitored with confocal microscopy and immunochemical detection of the expressed proteins. The results of these studies will lead to a better understanding of beaded filament function and the etiology of some forms of cataract.